Tyrosinase concentrate and extractant and method for making same



United States Patent TYROSINASE coNcnNrnatrnimsnnxrnacmNr AND METHODRoma-KING SAME y Erwin M. Cohen and LouisL. Lerner, Chicago, as-

signors to The Gillette Company, Boston, Mass, a corporation'of:Delaware.- i H This invention relatesto the preparation of a tyrosina'seconcentrate :and :pe'rtains more specifically t to a processfor thepreparation of a tyrosinase concentrate I frorna naturally occurringsource.

Still/another objectisto provide an improved extrac tant for a naturallyoccurring tyrosinase which IIIlIIi-r mizes discoloration: and"degradation of the tyrosinase during. the extraction; and. concentrationprocess;

A-: further: objectzis ttr providesa; simple; and. readilycontrolledvextraction process for;producinga high ac tivity tyrosinaseconcentrate having:-azdecreased-content ofcolor impurities,

Other and further objects. will be apparent from-the description'whichfollows.

Tyrosinase, an enzyme wh'ich is effective for produc-' ing melaninpigments,.is"-anaturally occurringenyzme which has been isolated from a.numberof ildiiferent sources; The naturally occurring material which isgenerally regarded 381 being" the. best-and: most convenient source.forthis enzyme isa mushroom (Psalliota campes= iris), althoughthe.enzyme may alsobe obtained from Idaho potatoes, yams, cuttlefish inksac, andffromaa'rtm roped.- blood. In: general the proced'ureafbr:preparing the :enzyme concentrate-from: any of these sourcesis toextract the enzyme from the naturally occurring source materialby meansof an aqueous extractant medium in whichwater may be the, sole liquidsolvent. or which may? contain a mixture of; water and acetone or waterand alcohol as the-liquid. solventi The term aqueous solution or aqueousextractant as. used hereinafter is intended to include any liquidsolvent or solvent mixturecontaining more than 5.0% by weight ofwater.

Although; a considerable amount ofwork has been done in an effort toimprovethe procedure-and obtain axtyrosinase concentrate of better'quality, the-re has remained the'basic difiiculty that the tyrosinase issubject to oxidation by the air of theatmosphere. Concentrates whichhave heretofore been obtained have'been dark colored: because of thepresence: of pigments" formed in. situ during the extractionpprocedureand have-been low in activity; for example, one firm markets aconcentrate having an activity of the orderiofl catecholase unit permilligram of. dry concentrate. This isrnostly due:- to the decompositionor deterioration of the tyro sinase during the extraction-procedure.Despite many attemptsto. increase, the: yield of tyrosinase from asource suchas mushrooms, for-example by the preliminary steps of.freezing and ithawing; mushroom pulp prior: to extraction, these; stepshave; failed; to provide any. substantial improvement in'color. oractivity (purity) of the final concentrate.

It has nowjbe'en' discovered 'thatby employfing as the ext ractant anaqueous. solutionv containing dissolved therein a reducing agent for.orthoquinones together with a? competitive inhibitorfor'j tyrosinase,;not onlyare the yield and aetivity of the concentrate greatly increased,but in addition the-procedure? for the'ext'raction may besimplified'andsliortenedl. Although a numberof different reducing;agents for orthoquinones are'known; any one'ormore of which may beemployed irr'th'e present invention, best' resultshave-beenobtained-with ascorbic acid and with hydroquinone: CtjenzymeHin ite-reduced form is also an effectivereducingfagent fororth'oquinones, but its cost arthpresentftime is soliigh that-its use isnot practical fronr an-'--economicpoint of-view;' It has" been foundthat-"an aciueous-extraetant containing as little as 0.5% by weight ofreducing agent is eifectiveforj the purposes of theapresent invention'r-While larger quantities may be employed up' tothe point at which-theaqueous solution becomes saturated 'with -the reducing agent (up to3'0'%'-by weight-inthe case of ascorbic acid), the use of such' largequantities isnot necessary to achieve the desired results: Ingenera'lj-it hasbeen found that an amount of reducingagentfrom 1% to 5%by weight of the aqueous extractant solution-provides best results.

The -competitive inhibitor of tyrosinase which is employed incombination=-with-theireducing agent in the aqueousext'raetant 'solutionmay be any one or more of the-conventional competitive inhibitors, amongwhich are benzoic: acid, o-chlorobenzoic acid, o-hyd'roxybenzoic acid;o-methoxybenzoic acid, o to-luic' acid, l-cin namic; acid,methylbenzoate, methyl salicyl'ate', acetophenone, nicotinicacid,cyclohexane carboxylic acid, trimethyl acetic acid, and the like; Ofthese benzoic acid has been found to b'e-mo-st effective and ispreferred. Whileiza's" little -as 0.02% by weight of competitiveinhibiton'basedi onrtlie total weight of aqueous extractant solution maybe employed, it is generally desirable to employ a saturatedsolutionwhieh contains, in the case of benzoic acid, about 0.2 byweight,based on the total. weight of the-aqueous extractant solution.

' Int: carryingout" the pro'cessof the present invention, the'i aqueousextractant solution containing dissolved therein; the" desired reducing:agent for orthoquinones together'iwith a competitive inhibitor oftyrosinase is employed to ext-ract th'e source material in the usualmanner while maintainingthe temperature close to 0 C. (from -5 C to +1 00.). The pH of the extractant'is'preferably maintainedfrom 5.2 to 5.5, asmall amount 2. of dilute: ammonium hydroxide being employed to adjustthei-pHfto a: value within the desired range when: necessary; The sourcematerial may be previously crushed'or chopped into small pieces in orderto ensure thorough extraction, audit is" also desirable in many casestosubject Ih8.-.SOl1IC6E material to further grinding, maceration orattrition during the extraction step. The amount of extractant solutionemployed is a matter of choice; the-greater. the ratioof extra'ctant tosource-material, the more thorough the extraction will be, but the moreliquid musb-beremovedt to achieve the final dry concentrate.

After separation of. the aqueous extract from the source material-in aconventional filtering operation, the source materialmay-'besubject'edto" one or more addi tional. extractions with: afreshbatch of aqueous extractant. The'extractant solution containing thetyrosinase is-then treatedeither with: a salt'solution or with acetonein order to: precipitate: the tyrosinase; wane a number ofdifferent-:water-soluble salts containing polyvalentianions may beemployed for precipitating" or 9 U salting out" the tyrosinase, bestresults have been obtainedwith ammonium sulfate, the extrac'tantsolu'tion""" preferably being made 0.8 saturated with the ammoniumsulfate. When acetoneis employed as the precipitant, best results areObtained by employing an amount of acetone which is from 1.2 to 1.7times the volume of the aqueous extractant solution. In either case theextractant solution should be maintained at a tlow tem-g perature duringthe precipitation step, which requires from 10 to 60 minutes forcompletion, the temperature preferably being maintained below 10 C., andin the case where acetone is employed as the precipitant, beingpreferably maintained below: -10 C..

filtering step. In order to obtain a product of high activity, the stepsof dissolving the tyrosinase in aqueous extractant solution andprecipitating it therefrom may be repeated as many times as desireduntil no further increase in activity of the product is attained. Theprecipitation step serves to separate the tyrosinase from the reducingagent and competitive inhibitor which tend to remain in solution.

When a salt such as ammonium sulfate has been employed as theprecipitant, the precipitate is then dissolved in water and subjected todialysis to remove anycoprecipitated salt, whereupon it may belyophilized (freezedried). 'When acetone has been employed as theprecipitant, no dialysis step is required, the precipitate simply beingdissolved in water and subjected to final concentration by aconventional lyophilization step. It is important that thelyophilization step be as complete as possible to provide a dry, stableproduct.

The process described in Example 1 is carried out up i, to the point ofaddition of ammonium sulfate. In place The resulting preclpitate 1scollected by a conventional of the ammonium sulfate there is added anamount of acetone equal to 1.5 times the volume of the aqueous solution,and the mixture is allowed to stand to permit precipitation to occurwhile maintaining the temperature at 7- -10" C. or lower. Theprecipitate is separated from thesolution by filtration, washed with amixture of acetone and water (3:2 by volume) at -10 C., and thendissolved in a few liters of cold (0 C.) deionized Water." Theprecipitation by means of acetone is repeated until the activity of theprecipitate shows no further increase. Usually two repetitions of theprecipitation sufiice. The precipitate is then dissolved in colddeionized water and lyophilized to produce a dry solid product verylight tan in appearance having an activity of 100 catecholase units permilligram.

The product of the present invention prepared as described above has anactivity of at least 10 catecholase units per milligram of dryconcentrate in the case of the product made using a salt precipitant. Inthe case of the product made by using acetone as the precipitant, theproduct has an activity of at least 80 catecholase units per milligramof dry concentrate.

In order to illustrate more clearly the nature of the present invention,the following specific examples are described without any intention tolimit the present invention to these examples.

Example 1 drained, and the mushrooms are then ground in a Waring Blendorwith 8 liters of deionized water containing 2 liters of crushed ice and1% by weight of ascorbic acid together with 0.2% by weight of benzoicacid. The pH of the solution is adjusted to 5.5 with a small amount ofdilute ammonium hydroxide, the pH being maintained between 5.2 and 5.5throughout the extraction step. After completion of the grinding, themixture is stirred gently for about an hour while maintaining thetemperature at approximately 0 C. to permit extraction of the tyrosinasefrom the mushrooms. time the mixture is filtered, the residue beingextracted in the filter with an additional quantity of approximately 3liters of extractant consisting of an aqueous solution containing 1% ofascorbic acid and 0.2% of benzoic acid It will be noted that whenacetone is used as the precipitant, the dialysis step may be omittedsince there is no salt to. be precipitated or occluded with thetyrosinase precipitate.

. Similar results are obtained when there is employed as the extractantsolution a mixture of water with 30% by weightof acetone, in whichmixture are dissolved the orthoquinone reducing agent and thecompetitive inhibitor of tyrosinase.

Although specific embodiments of the invention have been describedherein, it is not intended to limit the invention solely thereto, but toinclude all of the obvious variations and modifications within thespirit and scope of the appended claims. I

What is claimed is:

11. In the process of preparing a tyrosinase concentrate by extractionfrom a naturally occurring source at a temperature from 5' to +10 C.,the step which comprises employing as the extractant an aqueous solutioncontaining at least 0.5% by Weight of a member of the class consistingof ascorbic acid and hydroquinone, and at least 0.1% by weight of acompetitive inhibitor of tyrosinase. 2. In the process of preparing atyrosinase concentrate l by extraction from a naturally occurring sourceat a At the end of this {IL at pH 5.5. The two filtrates are combinedand sufiicient hours,-during which time a solid precipitate separates I'5 and in large part floats to the top of the dense solution. At the endof this time the mixture is filtered and the solid material is washedwith a cold aqueous solution containing ammonium sulfate 0.8 saturated.The solid material thus separated is dissolved in. cold deionized water(approximately 0-5 C.), placed in ,a sausage temperature from -5 to +10C., the step which comprises employing as the extractant an aqueoussolution containing at least 0.5 by weight of ascorbic acid and at least0.1% by weight of benzoic acid.

3. In the process of preparing a tyrosinase concentrate by extractionfrom a naturally occurring source at a temperature from -5 to +10 C.,the steps which comprise employing as the extractant an aqueous solutioncontaining at least 0.5% by weight of a member of the class consistingof ascorbic acid and hydroquinone and at least 0.1% by weight of acompetitive inhibitor of tyrosinase, and adding ammonium sulfate to saidextractant solution to precipitate a tyrosinase-rich material therefrom.4. The process defined in claim 3 comprising in addition the steps offorming an aqueous solution of said tyrosinase-rich material andsubjecting said solution to dialysis and then to lyophilization.

5. In the process of preparing a tyrosinase concentrate by extractionfrom mushrooms at a temperature from to +10 C., the steps which compriseemploying ca ing (Visiting), and. dialyzed againstcold deionized 75 astheextractantau aqueous solution containing at least 0.5% by weight of amember of the class consisting of ascorbic acid and hydroquinone and atleast 0.1% by weight of a competitive inhibitor of tyrosinase, and thenadding acetone to said extractant solution to precipitate atyrosinase-rich material.

6. The process defined in claim 5 comprising in addition the steps offorming an aqueous solution of said tyrosinase-rich material andsubjecting said solution to lyophilization.

7. In the process of preparing a tyrosinase concentrate by extractionfrom mushrooms at a temperature from 5 to +10 C., the steps whichcomprise employing as the extractant an aqueous solution containing atleast 0.5% by weight of ascorbic acid and at least 0.1% by Weight ofbenzoic acid, adding acetone to said extractant solution to precipitatea tyrosinase-rich material, forming an aqueous solution of saidtyrosinase-rich material, and subjecting said solution to lyophilizationto provide a dry solid tyrosinase concentrate.

8. An extractant for use in preparing a tyrosinase concentrate from anaturally occurring source which comprises an aqueous solutioncontaining dissolved therein at least 0.5% by weight of a member of theclass consisting of ascorbic acid and hydroquinone, and at least 0.1% byweight of a competitive inhibitor of tyrosinase.

9. An eXtractan-t for use in preparing a tyrosinase concentrate from anaturally occurring source which comprises an aqueous solutioncontaining dissolved therein at least 0.5% by weight of ascorbic acidand at least 0.1% by weight of benzoic acid.

References Cited in the file of this patent Advances In Enzymology byNord et al., Intersoience Publishers Inc., New York (1944), vol. 4, pp.101, 102, 116, 117, 121 and 122.

Chemistry and Methods of Enzymes by Sumner et al., Academic Press Inc.,1953, New York, pp. 240 to 243.

Methods In Enzymology by Colowick et al., Academic Press Inc., New York,1955, vol. 2, pp. 822 to 827.

2. IN THE PROCESS OF PREPARING A TYROSINASE CONCENTRATE BY EXTRACTIONFROM A NATURALLY OCCURRING SOURCE AT A TEMPERATURE FROM -5* TO + C., THESTEP WHICH COMPRISES EMPLOYING AS THE EXTRACTANT AN AQUEOUS SOLUTIONCONTAINING AT LEAST 0.5% BY WEIGHT OF ASCORBIC ACID AND AT LEAST 0.1% BYWEIGHT OF BENZOIC ACID.